The specific aims of this proposal are to identify and characterize some of the genetic determinants of pathogenicity in Campylobacter. The major goals we accomplished in the first proposal were to develop methods to permit genetic exchange that would allow us to clone putative virulence determinants and to construct isogenic mutants. We have successfully completed these goals by developing shuttle plasmids that can be introduced into Campylobacter via bacterial conjugation and by transformation via high voltage electroporation. Therefore, we can now proceed to establish genetic libraries in Campylobacter for the first time. Among the determinants of pathogenicity that we will examine are those genes encoding for Campylobacter flagellin, cytotoxin, and LPS in C. jejuni and surface proteins mediating serum resistance in C. fetus. While the primary focus will be on Campylobacter flagellin and cytotoxin, we will also examine the others, in part as a means to establish the utility of the two genetic exchange methods. Using shuttle vectors developed in the laboratory we will establish isogenic mutants deficient in these factors via gene exchange and transposon insertion. By examining the behavior of isogenic mutants in relevant models of infection, and by employing cloned pathogenic determinants as probes, we will be able to more precisely assess the contribution of each factor in the pathogenesis of gastrointestinal tract infection.